A. Field of the Invention
This invention relates generally to photonic crystal biochemical sensor devices. Such devices are used for optical detection of the adsorption of a biological material, such as DNA, protein, viruses or cells, or chemicals, onto a surface of the device or within a volume of the device. More particularly, this invention is related to a biosensor having the form of a photonic crystal having defect cavities formed in a periodic pattern in the device. The invention provides a higher sensitivity and a greater degree of spatial localization of incoupled photons than previously reported photonic crystal biosensor devices.
B. Description of Related Art
Photonic Crystals
Photonic crystals represent a new class of optical devices that have been enabled by recent advances in semiconductor fabrication tools with the ability to accurately deposit and etch materials with precision less than 100 nm. Photonic crystals are characterized by an infinite or semi-infinite periodic structure containing alternating materials of low dielectric permittivity and high dielectric permittivity. In principle, a photonic crystal structure may extend in 1, 2, or 3 dimensions of space. For background information on photonic crystals, the reader is directed to Joannopoulos, J. D., R. D. Meade, and J. N. Winn, Photonic Crystals, 1995 Princeton, N.J.: Princeton University Press.
Along with the development of appropriate fabrication methods, accurate computer modeling tools are also becoming available which facilitate design of components with the ability to manipulate the propagation of light within a photonic crystal structure. Like the periodic arrangement of atoms within a semiconductor crystal that results in the formation of energy bands which dictate the conduction properties of electrons, the periodic arrangement of macroscopic dielectric media within a photonic crystal is designed to control the propagation of electromagnetic waves. Because the period of the structure is smaller than the wavelength of light, such devices are often referred to as “sub-wavelength surfaces” or as “nanostructured surfaces” because typical dimensions are 50–300 nm. Using photonic crystal design principles, one may construct devices with optical energy bands, which effectively prevent the propagation of light in specified directions and energies, while allowing concentration of electromagnetic field intensity within desired volumes and surfaces. See, e.g., Munk, B. A., Frequency Selective Surfaces. Wiley Interscience. 2000: John Wiley & Sons; Pacradouni, V., W. J. Mandeville, A. R. Cowan, P. Paddon, J. F. Young, and S. R. Johnson, Photonic band structure of dielectric membranes periodically textured in two dimensions. Physical Review B, 2000. 62(7): p. 4204–4207.
The applications of photonic crystal structures within the field of optoelectronics have been numerous, including integration with lasers to inhibit or enhance spontaneous emission, waveguide angle steering devices, and narrowband optical filters. See e.g. Quang, T., M. Woldeyohannes, S. John, and G. S. Agarwal, Coherent control of spontaneous emission. Physical Review Letters, 1997. 79(26): p. 5238–5241 Liu, Z. S., S. Tibuleac, D. Shin, P. P. Young, and R. Magnusson, High efficiency guided-mode resonance filter. Optics Letters, 1998. 23(19): p. 1556–1558; Peng, S., Experimental demonstration of resonant anomalies in diffraction from two-dimensional gratings. Optics Letters, G. Michael Morris. 21(8): p. 549–551; Magnusson, R. and S. S. Wang, New principle for optical filters. Applied Physics Letters, 1992. 61(9): p. 1022–1024. Several device applications take advantage of the photonic crystal structure geometry's capability for concentrating light into extremely small volumes with very high local electromagnetic field intensity.
Defect cavity photonic crystals have been widely reported in the literature for their ability to enhance the Q and to spatially localize regions of high electromagnetic field intensity. John, S., Strong localization of photons in certain disordered dielectric superlattices. Physical Review Letters, 1987. 58(23): p. 2486–2489; Scherer, A., T. Yoshie, M. Loncar, J. Vuckovic, K. Okamoto, and D. Deppe, Photonic crystal nanocavities for efficient light confinement and emission. Journal of the Korean Physical Society, 2003. 42: p. 768–773; Srinvasan, K., P. E. Barclay, O. Painter, J. Chen, A. Y. Cho, and C. Gmachi, Experimental demonstration of a high quality factor photonic crystal microcavity. Applied Physics Letters, 2003. 83(10): p. 1915–1917; Painter, O., K. Srinivasan, J. D. O'Brien, A. Scherer, and P. D. Dapkus, Tailoring of the resonant mode properties of optical nanocavities in two-dimensional photonic crystal slab waveguides. Journal of Optics A: Pure and Applied Optics, 2001. 3: p. S161–S170 and John, S. and V. I. Rupasov, Multiphoton localization and propagating quantum gap solitons in a frequency gap medium. Physical Review Letters, 1997. 79(5): p. 821–824. Periodic arrays of defect cavities in a photonic crystal are reported in Altug, H. and J. Vuckovic, Two-dimensional coupled photonic crystal resonator arrays. Applied Physics Letters, 2004. 84(2): p. 161–163.
Photonic Crystal Biosensors
Several properties of photonic crystals make them ideal candidates for application as optical biosensors. First, the reflectance/transmittance behavior of a photonic crystal can be readily manipulated by the adsorption of biological material such as proteins, DNA, cells, virus particles, and bacteria. Each of these types of material has demonstrated the ability to alter the optical path length of light passing through them by virtue of their finite dielectric permittivity. Second, the reflected/transmitted spectra of photonic crystals can be extremely narrow, enabling high-resolution determination of shifts in their optical properties due to biochemical binding while using simple illumination and detection apparatus. Third, photonic crystal structures can be designed to highly localize electromagnetic field propagation, so that a single photonic crystal surface can be used to support, in parallel, the measurement of a large number of biochemical binding events without optical interference between neighboring regions within <3–5 microns. Finally, a wide range of materials and fabrication methods can be employed to build practical photonic crystal devices with high surface/volume ratios, and the capability for concentrating the electromagnetic field intensity in regions in contact with a biochemical test sample. The materials and fabrication methods can be selected to optimize high-volume manufacturing using plastic-based materials or high-sensitivity performance using semiconductor materials.
Representative examples of biosensors in the prior art are disclosed in Cunningham, B. T., P. Li, B. Lin, and J. Pepper, Colorimetric resonant reflection as a direct biochemical assay technique. Sensors and Actuators B, 2002. 81: p. 316–328; Cunningham, B. T., J. Qiu, P. Li, J. Pepper, and B. Hugh, A plastic calorimetric resonant optical biosensor for multiparallel detection of label-free biochemical interactions, Sensors and Actuators B, 2002. 85: p. 219–226; Haes, A. J. and R. P. V. Duyne, A Nanoscale Optical Biosensor: Sensitivity and Selectivity of an Approach Based on the Localized Surface Plasmon Resonance Spectroscopy of Triangular Silver Nanoparticles. Journal of the American Chemical Society, 2002. 124: p. 10596–10604.
The combined advantages of photonic crystal biosensors may not be exceeded by any other label-free biosensor technique. The development of highly sensitive, miniature, low cost, highly parallel biosensors and simple, miniature, and rugged readout instrumentation will enable biosensors to be applied in the fields of pharmaceutical discovery, diagnostic testing, environmental testing, and food safety in applications that have not been economically feasible in the past.
In order to adapt a photonic bandgap device to perform as a biosensor, some portion of the structure must be in contact with a liquid test sample. Biomolecules, cells, proteins, or other substances are introduced to the portion of the photonic crystal and adsorbed where the locally confined electromagnetic field intensity is greatest. As a result, the resonant coupling of light into the crystal is modified, and the reflected/transmitted output (i.e., peak wavelength) is tuned, i.e., shifted. The amount of shift in the reflected output is related to the amount of substance present on the sensor. The sensors are used in conjunction with an illumination and detection instrument that directs polarized light into the sensor and captures the reflected or transmitted light. The reflected or transmitted light is fed to a spectrometer that measures the shift in the peak wavelength.
The ability of photonic crystals to provide high quality factor (Q) resonant light coupling, high electromagnetic energy density, and tight optical confinement can also be exploited to produce highly sensitive biochemical sensors. Here, Q is a measure of the sharpness of the peak wavelength at the resonant frequency. Photonic crystal biosensors are designed to allow a liquid test sample to penetrate the periodic lattice, and to tune the resonant optical coupling condition through modification of the surface dielectric constant of the crystal through the attachment of biomolecules or cells. Due to the high Q of the resonance, and the strong interaction of coupled electromagnetic fields with surface-bound materials, several of the highest sensitivity biosensor devices reported are derived from photonic crystals. See the Cunningham et al. papers cited previously. Such devices have demonstrated the capability for detecting molecules with molecular weights less than 200 Daltons (Da) with high signal-to-noise margins, and for detecting individual cells. Because resonantly-coupled light within a photonic crystal can be effectively spatially confined, a photonic crystal surface is capable of supporting large numbers of simultaneous biochemical assays in an array format, where neighboring regions within ˜10 μm of each other can be measured independently. See Li, P., B. Lin, J. Gerstenmaier, and B. T. Cunningham, A new method for label-free imaging of biomolecular interactions. Sensors and Actuators B, 2003.
There are many practical benefits for biosensors based on photonic crystal structures. Direct detection of biochemical and cellular binding without the use of a fluorophore, radioligand or secondary reporter removes experimental uncertainty induced by the effect of the label on molecular conformation, blocking of active binding epitopes, steric hindrance, inaccessibility of the labeling site, or the inability to find an appropriate label that functions equivalently for all molecules in an experiment. Label-free detection methods greatly simplify the time and effort required for assay development, while removing experimental artifacts from quenching, shelf life, and background fluorescence. Compared to other label-free optical biosensors, photonic crystals are easily queried by simply illuminating at normal incidence with a broadband light source (such as a light bulb or LED) and measuring shifts in the reflected color. The simple excitation/readout scheme enables low cost, miniature, robust systems that are suitable for use in laboratory instruments as well as portable handheld systems for point-of-care medical diagnostics and environmental monitoring. Because the photonic crystal itself consumes no power, the devices are easily embedded within a variety of liquid or gas sampling systems, or deployed in the context of an optical network where a single illumination/detection base station can track the status of thousands of sensors within a building. While photonic crystal biosensors can be fabricated using a wide variety of materials and methods, high sensitivity structures have been demonstrated using plastic-based processes that can be performed on continuous sheets of film. Plastic-based designs and manufacturing methods will enable photonic crystal biosensors to be used in applications where low cost/assay is required, that have not been previously economically feasible for other optical biosensors.
The assignee of the present invention has developed a first generation photonic crystal biosensor and associated detection instrument. The sensor and detection instrument are described in the patent literature; see U.S. patent application publications U.S. 2003/0027327; 2002/0127565, 2003/0059855 and 2003/0032039. Methods for detection of a shift in the resonant peak wavelength are taught in U.S. Patent application publication 2003/0077660. The biosensor described in these references include 1- and 2-dimensional periodic structured surfaces produced on continuous sheets of plastic film. The crystal resonant wavelength is determined by measuring the peak reflectivity at normal incidence with a spectrometer to obtain a wavelength resolution of 0.5 picometer. The resulting mass detection sensitivity of <1 pg/mm2 (obtained without 3-dimensional hydrogel surface chemistry) has not been demonstrated by any other commercially available biosensor.
A fundamental advantage of first-generation photonic crystal biosensor devices is their ability to be mass-manufactured with plastic materials in continuous processes at a 1–2 feet/minute rate. Methods of mass production of the sensors are disclosed in U.S. Patent application publication 2003/0017581. As shown in FIG. 1, the periodic surface structure of a biosensor 10 is fabricated from a low refractive index material 12 that is overcoated with a thin film of higher refractive index material 14. The low refractive index material 12 is bonded to a substrate 16. The surface structure is replicated within a layer of cured epoxy 12 from a silicon-wafer “master” mold (i.e. a negative of the desired replicated structure) using a continuous-film process on a polyester substrate 16. The liquid epoxy 12 conforms to the shape of the master grating, and is subsequently cured by exposure to ultraviolet light. The cured epoxy 12 preferentially adheres to the polyester substrate sheet 16, and is peeled away from the silicon wafer. Sensor fabrication was completed by sputter deposition of 120 nm titanium oxide (TiO2) high index of refraction material 14 on the cured epoxy 12 grating surface. Following titanium oxide deposition, 3×5-inch microplate sections were cut from the sensor sheet, and attached to the bottoms of bottomless 96-well and 384-well microtiter plates with epoxy.
As shown in FIG. 2, the wells 20 defining the wells of the mircotiter plate contain a liquid sample 22. The combination of the bottomless microplate and the biosensor structure 10 is collectively shown as biosensor apparatus 26. Using this approach, photonic crystal sensors are mass produced on a square-yardage basis at very low cost.
The first-generation detection instrument for the photonic crystal biosensor is simple, inexpensive, low power, and robust. A schematic diagram of the system is shown in FIG. 2. In order to detect the reflected resonance, a white light source illuminates a ˜1 mm diameter region of the sensor surface through a 100 micrometer diameter fiber optic 32 and a collimating lens 34 at nominally normal incidence through the bottom of the microplate. A detection fiber 36 is bundled with the illumination fiber 32 for gathering reflected light for analysis with a spectrometer 38. A series of 8 illumination/detection heads 40 are arranged in a linear fashion, so that reflection spectra are gathered from all 8 wells in a microplate column at once. See FIG. 3. The microplate+biosensor 10 sits upon a X-Y addressable motion stage (not shown in FIG. 2) so that each column of wells in the microplate can be addressed in sequence. The instrument measures all 96 wells in ˜15 seconds, limited by the rate of the motion stage. Further details on the construction of the system of FIGS. 2 and 3 are set forth in the published U.S. patent application Ser. No. 2003/0059855.